2021 Volume 44 Issue 2 Pages 266-270
Extracellular pH (pHe) of tumor cells is characteristic of tumor microenvironment (TME). Acidic TME impairs the responses of tumors to some anti-cancer chemotherapies. In this study, we showed that daily oral dosing of sodium potassium citrate (K/Na citrate) increased blood HCO3− concentrations, corresponding to increase of HCO3− concentrations and pHs in urine, and neutralized the tumor pHe. Neutralization of acidic TME by alkaline substance like HCO3−, an active metabolite of K/Na citrate, well potentiated the therapeutic effect of anticancer agent TS-1®, an orally active 5-fuluoro-uracil derivative, in Panc-1 pancreatic cancer-xenograft murine model. Neutralization of acidic TME by using an alkaline K/Na citrate is a smart approach for enhancement of the therapeutic effects of anticancer agents for pancreatic cancer in the end stage.
Several reports showed that the interstitial extracellular pH (pHe) in tumor is acidic (pH 6.2–6.9) rather than normal tissues (pH 7.3–7.4).1,2) This is caused in part of the overstimulation of several ion transporters such as Na+/H+ exchanger (NHE-1), Na+-dependent and independent HCO3−/Cl− exchangers and the mono-carboxylate transporter (MCT), which increase H+ ions in extracellular space and acidify the pHe in tumors.3) In addition, the Warburg effect, a tumor-associating anabolic glycolysis, is one of the principal factors inducing acidic tumor microenvironment (TME) in the tumor extracellular region.4) It was reported that the acidic TME is relating to tumor progression and metastasis.5,6)
The acidic TME is relating to tumor responses to anti-cancer chemotherapeutic agent, which results in aggressive phenotype of tumors characterized by chemoresistance7) and suppress apoptosis caused by activating nuclear factor-kappaB (NF-κB) and Bcl-2-associated X protein (BAX).8) The acidic TME also causes p53-dependent apoptosis and harbors p53-mutated cancer cells that lose the apoptotic potency and are not responsive to the apoptosis-inducing anticancer drug.9)
Alkaline product of sodium potassium citrate (K/Na citrate) has been used for clinical treatment of the metabolic acidosis in patients.10) In human body, K/Na citrate is metabolized to HCO3− in the intestine after oral dosing of K/Na citrate.11) The HCO3− buffer system plays an important role of maintaining the homeostatic pH in the blood through balancing of the composition of carbonic acid (H2CO3), HCO3− and carbon dioxide (CO2), as consequence, contributes to modulation of tumor pHe.12) It is assumed to be increased serum HCO3− concentrations and deliver excess HCO3− into the tumors after oral administration of K/Na citrate. The HCO3− would trap an H+ ion and forms H2CO3 in the tumor interstitial space, resulting in the neutralization of tumor pHe. Renal filtration regulates blood levels of HCO3− through glomerular filtration and acid secretion.13)
TS-1® is an oral anticancer drug consisting of combinations of tegafur, 5-chloro-2,4-dihydroxypridine (CDHP) and potassium oxonate, having some clinical success in the treatment of patients with malignant cancers including pancreatic cancer.14,15) TS-1® is known to maintain therapeutic concentrations of 5-fluorouracil (5-FU) in blood and the tumor via metabolism of a prodrug tegafur in the TS-1®.16) We investigated the effect of oral administration of K/Na citrate on the serum HCO3− concentrations along with uric HCO3− concentrations and uric pHs, and on the tumor pHe. And the therapeutic effect of the combination of daily oral dosing of TS-1® with daily oral dosing of K/Na citrate in a Panc-1 human pancreatic cancer xenograft murine model.
K/Na citrate, a mixture composed of 2 mol potassium citrate, 2 mol sodium citrate and 1 mol citric acid hydrate was purchased from Nippon Chemiphar (Tokyo, Japan). TS-1® was obtained from Taiho Pharmaceutical (Tokyo, Japan). All other reagents were of analytical grade.
Cells and AnimalsColon-26 murine colorectal carcinoma cell line (RCB2657) and Panc-1 human pancreatic carcinoma cell line (RCB2095) were purchased from the RIKEN BioResource Center (Ibaraki, Japan). The cells were cultured in RPMI-1640 medium (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) supplemented with 10% heat-inactivated fetal bovine serum (Corning, NY, U.S.A.), 100 units/mL penicillin and 100 µg/mL streptomycin (ICN Biomedicals, CA, U.S.A.) in a 5% CO2/air incubator at 37 °C.
BALB/c mice (female, 5 weeks old) and BALB/c nu/nu mice (female, 5 weeks old) were purchased from Japan SLC (Shizuoka, Japan). The experimental animals were allowed free access to water and mouse chow, and were housed under controlled environmental conditions (constant temperature and humidity, and a 12-h dark–light cycle). All animal experiments were evaluated and approved by the Animal and Ethics Review Committee of Tokushima University.
Tumor-Bearing MiceColon-26 tumor-bearing model was established by subcutaneous inoculation of Colon-26 cells (2 × 106 cells/mouse) at a flank region of BALB/c mice. Panc-1 tumor-bearing mouse model was established by subcutaneous inoculation of Panc-1 cells (5 × 106 cells/mouse) at a flank region of BALB/c nu/nu mice. All animal experiments were initiated when the tumors reached 50–100 mm3 in size.
Measurement of Serum HCO3−, Urine HCO3− and Urine pHBALB/c mice were orally administered with a dose of K/Na citrate (2500 mg/kg). At selected time points post administration (0, 0.5, 1, 2, 4, 6, 8, and 24 h), blood was collected from the postcaval vein of the mice. Serum samples were obtained by centrifugation of the blood (3000 rpm, 4 °C, 15 min) following by incubation for 30 min at room temperature. The serum HCO3− concentrations were measured using the HCO3− measurement kit (Diacolor® CO2 clinical diagnostic reagent, TOYOBO, Osaka, Japan). At the same time points, urine was collected by pushing on the lower abdominal region of the mice. The urine HCO3− concentrations were measured using Diacolor® CO2. Urine pH values were measured with the pH test paper (pH 5.5–9.0, AS ONE, Osaka, Japan).
Measurement of Intratumor pH Using a MicroelectrodeColon-26 tumor-bearing mice were orally administered with a dose of K/Na citrate (500 mg/kg) for 14 d. The interstitial pHe in the tumor was measured using microelectrode with pH meter as referred the previous publications.17,18) The mice were anesthetized by isoflurane inhalation and then stabbed with a reference electrode (MI-401F, Microelectrodes, NH, U.S.A.) into the subcutaneous region or with a pH electrode (MI-408B TIP, Microelectrodes) into the center of the tumor. Electrodes were calibrated before the measurements using standard pH 4.01 and 7.00 buffers. One measurement was taken at each mouse and plotted (n = 7–9).
Therapeutic Effects of Oral Administration of K/Na Citrate Combined with Oral Administration of TS-1® Treatment in a Panc-1-Xenograft Murine ModelPanc-1 tumor-bearing mice were orally administered with 14 daily doses of K/Na citrate (500 mg/kg/d) combined with TS-1® (18 mg/kg/d). Volume of tumors and body weight of the treated mice were recorded twice weekly. Tumor growth inhibition [TGI (%)] was calculated using the following formula (RTV: relative tumor volume).19)
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Statistical differences between the groups were evaluated by ANOVA with the Tukey post-hoc test using the Prism 8 software (GraphPad Software, San Diego, CA, U.S.A.). All values are reported as the mean ± standard deviation (S.D.). The levels of significance were set at * p < 0.05, *** p < 0.001.
It is known that oral K/Na citrate produces HCO3− in the body and helps to correct the acid buildup in the blood.11) The dosage of K/Na citrate was decided by reference to the previous publication.20) A single administration of K/Na citrate, given to naive mice, clearly increased serum HCO3− concentrations until 2 h post administration, and then gradually decreased down to base over 48 h (Fig. 1A). The administration of K/Na citrate rapidly increased HCO3− concentrations in urine by 1 h post administration and then decreased down to the base level until 6 h (Fig. 1B). In addition, the administration of K/Na citrate increased urine pH to approx. 8.5 within 2 h post administration and then fell back to pH 5.9 within 6 h (Fig. 1C), which is highly consistent with the change of urine HCO3− concentrations. These results indicate that an oral administration of K/Na citrate may increase the pH of extracellular fluid in vivo by increasing blood HCO3− concentrations.
BALB/c mice were orally administered with a dose of K/Na citrate (2500 mg/kg). At selected time points post administration (0, 0.5, 1, 2, 4, 6, 8, and 24 h), blood and urine were collected from the treated mice. (A) Serum HCO3− concentrations, (B) urine HCO3− concentrations, and (C) Urine pHs were determined. The data are means ± standard deviation (S.D.) (n = 5–6).
As shown in Fig. 2A, pHs in subcutaneous region or intratumor region of Colon-26 tumors were directly measured with pH electrode probes as described in several literatures.17,18) Subcutaneous pH in normal mice, as a reference, was neutral (pH 7.15 ± 0.07) (Fig. 2B). The tumor pHe in non-treated mice was acidic (pH 6.67 ± 0.08), which is consistent with the other observations.17,21) Meanwhile, after treatment with K/Na citrate, the tumor pHe was clearly increased up to pH 6.90 ± 0.11, compared to that in non-treatment mice. These results confirm that daily administrations of K/Na citrate increase the pH of extracellular fluid of solid tumors.
Colon-26 tumor-bearing mice were orally administered with a dose of K/Na citrate (500 mg/kg) for 14 d. Then, the interstitial pHe in the tumor was measured using microelectrode with pH meter. (A) A reference electrode and a pH electrode were stabbed into the subcutaneous region and the center of tumor, respectively. (B) One measurement was taken at each mouse and plotted as a violin plot (n = 7–9). *** p < 0.001 vs. subcutaneous, ###p < 0.001 vs. intratumor (control). (Color figure can be accessed in the online version.)
Effect of neutralization of tumor pHe by oral K/Na citrate on in vivo tumor growth was investigated when combined with TS-1®. Neither K/Na citrate alone nor TS-1® inhibited the growth of tumor under our experimental condition. Meanwhile, the combined treatment showed suppression of the growth of Panc-1 tumor (Fig. 3A). These indicate that neutralization of tumor pHe by K/Na citrate administration increased the therapeutic effect of TS-1®, which did not show any therapeutic effect by mono-treatment. In the K/Na citrate treatment group (mono-treatment or combined treatment), any significant body weight changes were observed (Fig. 3B), indicating that the 14 d the K/Na citrate treatment was entirely tolerable.
Panc-1 tumor-bearing mice were orally administered with TS-1® (18 mg/kg/d, daily) for 2 weeks, combined with K/Na citrate (500 mg/kg/d, daily). (A) Tumor volumes and (B) body weights of mice were recorded twice weekly. The data are means ± S.D. (n = 8, * p < 0.05 vs. control).
| Treatment | TGI (%) | RTV |
|---|---|---|
| Control | — | 9.5 ± 0.9 |
| K/Na citrate | 13.4 | 8.3 ± 0.6 |
| TS-1® | 14.9 | 8.1 ± 0.7 |
| Combination | 32.4 | 6.4 ± 0.3* |
* p < 0.05 vs. control.
In the current study, we showed an oral administration of K/Na citrate increases serum HCO3− concentrations, corresponding to urine HCO3− concentrations and urine pHs (Fig. 1). In addition, we showed that chronical oral administrations of K/Na citrate (14 d) successfully neutralize the tumor pHe (Fig. 2). It has been reported that acidic TME can impair the responses to anticancer chemotherapy.22) One can hypothesize that therapeutic interventions designed for increasing tumor pHe may be able to improve therapeutic outcomes of some anticancer agents. We tested this hypothesis using Panc-1 xenograft murine model and TS-1® that is used for advanced pancreatic cancer in Japan.23) It was reported that the tumor acidity facilitates the epithelial mesenchymal transition (EMT) of pancreas cancer,24) which plays a role in the development of drug resistance against a cytotoxic chemotherapy including 5-FU, an active pharmaceutical ingredient in TS-1®, via over expression of Zeb-1, a transcriptional factor, and poor expression of E-cadherin, a cell adhesion protein.25) We confirmed that increasing tumor pHe by oral K/Na citrate potentiates the therapeutic efficacy of TS-1® without any systemic adverse effects (Fig. 3). Recently, the Japanese National Cancer Center reported that the 3-year survival rate of the pancreatic cancer patients with end stages at stage III and stage IV was 11.9 and 2.5%, respectively.23,24,26) In clinical cases, it must be difficult to treat with a full dose of anti-cancer agent, such as TS-1®, for pancreatic cancer patients with the end stage. In the present study, we newly investigated that the therapeutic effect of a decreased dose of TS-1®, that showed no effects by mono-treatment, was potentiated under the condition of TME neutralized by alkaline K/Na citrate on Panc-1 pancreatic cancer xenograft model.
Orally administered K/Na citrate is absorbed from the intestinal tract into blood and dissociated into its constituent ions. The citrate anion is excreted through the urine, causing a shift in the electrical equilibrium.27) In order to recover this homeostasis, blood HCO3− concentrations increase accompanied with a decrease of serum H+ ions,11) corresponding to decreasing H+ ions in tumor tissue. Accordingly, oral K/Na citrate can produce HCO3− appearing in, or disappearing from, the blood (Fig. 1A). It is well known that excess HCO3− in the blood is eliminated through glomerular filtration into urine; renal filtration regulates blood levels of HCO3− and acid secretion.13) Subsequently, urine HCO3− concentrations and urine pHs were increased in conjunction with increases in serum HCO3− concentrations. Interestingly, urine HCO3− concentrations and pHs rapidly dropped to base levels (Figs. 1B, C). This might be due to the presence of excess citrate ions secreted in urine that would tend to acidify the urine.
In recent days, there have been several clinical reports suggesting that internal acid-base balances are important for the health status of patients and may be involved in the therapeutic outcomes. In colorectal cancer patients undergoing resection of their primary tumors, the group with low serum HCO3− concentrations showed a lower 30-d overall survival than the group with normal HCO3− levels.28) In addition, in advanced pancreatic cancer patients treated with an alkaline diet with supplementary HCO3−, the median overall survival was significantly longer in the patients with alkaline uric pH (>7.0) than those with acidic uric pH (≤ 7.0) (16.1 and 4.7 months, respectively; p < 0.05).29) The neutralization of acidic TME via increasing serum HCO3− concentrations by oral chronic K/Na citrate might make patients better in physical condition and subsequently prevent the malignant tumor growth.
In conclusion, we demonstrated that the treatment with K/Na citrate neutralizes the acidic tumor’s interstitial pHe through elevating serum HCO3− concentrations, corresponding to increased urine HCO3− concentrations and urine pHs. Increasing tumor pHe by oral K/Na citrate potentiates the therapeutic efficacy of TS-1® without any severe systemic adverse effects. Our results imply that K/Na citrate, when orally and chronically administered, improve therapeutic effect of some types of chemotherapeutic agents by modulating tumor pHe.
This study was supported in part by Japan Society for the Promotion of Science, Grant-in-Aid for Young Scientists (19K16415).
Kiyoshi Eshima is President at Delta-Fly Pharma, Inc. No potential conflicts of interest were disclosed by the other authors.
